SUMMARY: Progressive loss of retinal ganglion cells (RGCs) and subsequent degeneration of optic nerve is among the most common ophthalmic neuropathies that affect populations worldwide. A promising therapeutic strategy relies on using stem cells to reconstruct functional retinal ganglion cells in vitro that can be used to replace dying cells in affected patients. However, for this strategy to be successful, a thorough understanding to the molecular mechanisms of RGC specification and differentiation are needed to elucidate and exploit normal RGC developmental pathways and thereby maximize RGC generation for cell replacement therapies. Our previous work has mapped the epigenetic landscape dynamics during mouse and human developing retina. However, there is a fundamental gap in our knowledge to the role of 3D chromatin topology in RGC development and maintenance. Experiments in this proposal will address this role by combining innovative state of the art genomic and genetic tools to elucidate the chromatin architecture dynamics that accompany RGC genesis and to determine how they function in vivo. Our central hypothesis is that temporal and spatial regulation of ganglion cell genesis is associated with dynamic 3D genomic interactions between specific non-coding DNA elements and genes that drive RGC differentiation and optic nerve growth. To test this hypothesis, we will elucidate the link between 3D chromatin architecture and the expression of a transcription factor essential for RGC formation. We will also dissect the functional significance of constituents of the regulatory landscape that accommodates RGC differentiation in vivo. Finally, we will integrate ChIP-Seq data with techniques that map chromatin topology to elucidate the long-range genomic interactions that are associated with components of the regulatory networks that are required for RGC differentiation. When completed the results of this proposal will advance our understanding to the 3D regulatory landscape that accommodates the generation of RGCs in vivo, a necessary knowledge to enhance RGC replacement therapies in RGC degenerative diseases. Project Summary- Al Diri 1